Abstract
c-Fms is the macrophage colony-stimulating factor (M-CSF) receptor, and intracellular signalling via the M-CSF/c-Fms axis mediates both innate immunity and bone remodelling. M-CSF-induced transient proteolytic degradation of c-Fms modulates various biological functions, and protein kinase C (PKC) signalling is activated during this proteolytic process via an unknown mechanism. Notably, the role of specific PKC isoforms involved in c-Fms degradation during osteoclast differentiation is not known. Here, we observed that inactivation of PKCδ by the biochemical inhibitor rottlerin, a cell permeable peptide inhibitor, and short hairpin (sh) RNA suppresses osteoclast differentiation triggered by treatment with M-CSF and receptor activator of NF-κB ligand. Interestingly, inhibition of PKCδ by either inhibitor or gene silencing of PKCδ accelerated M-CSF-induced proteolytic degradation of membrane-bound c-Fms via both the lysosomal pathway and regulated intramembrane proteolysis (RIPping), but did not affect c-fms expression at the mRNA level. Degradation of c-Fms induced by PKCδ inactivation subsequently inhibited M-CSF-induced osteoclastogenic signals, such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice administered PKCδ inhibitors into the calvaria periosteum exhibited a decrease in both osteoclast formation on the calvarial bone surface and the calvarial bone marrow cavity, which reflects osteoclastic bone resorption activity. These data suggest that M-CSF-induced PKCδ activation maintains membrane-anchored c-Fms and allows the sequential cellular events of osteoclastogenic signalling, osteoclast formation, and osteoclastic bone resorption.
Highlights
Macrophage colony-stimulating factor (M-CSF) mediates the differentiation of monocytic cells into phagocytic mononuclear macrophages relevant to osteoclast precursors, and subsequently participates in their survival, proliferation, and phagocytic function[1]
We previously reported that macrophage colony-stimulating factor (M-CSF) is critical for osteoclast differentiation, and that it activates PKCα and PKCδ23
To determine the role of PKCα and PKCδ signalling in osteoclast differentiation, osteoclast precursors were differentiated into multinucleated osteoclasts in the presence of M-CSF and RANKL for 4 days
Summary
Macrophage colony-stimulating factor (M-CSF) mediates the differentiation of monocytic cells into phagocytic mononuclear macrophages relevant to osteoclast precursors, and subsequently participates in their survival, proliferation, and phagocytic function[1]. C-Fms that becomes dimerised in response to M-CSF is rapidly degraded via RIPping[13] This process is common for cell surface proteins, such as Fas and Fas ligand, IL-2 and IL-6 receptor, TNFα and receptor activator of NF-κB ligand (RANKL)[14]. Among the various PKC isoforms, PKCβ and PKCε are involved in the respective regulation of heparin-binding EGF-like growth factor and TNFα shedding[20,21] and PKCδ and PKCη are involved in regulating IL-6 receptor shedding[22] These results indicate that PKC signalling may act as a positive regulator of ectodomain shedding during RIPping. In contrast to previous reports, we propose that M-CSF-mediated PKCδ activation negatively regulates lysosomal- and RIPping-dependent proteolytic degradation of the membrane-bound M-CSF receptor c-Fms, thereby retarding c-Fms proteolytic degradation, sustaining M-CSF-induced osteoclastogenic signalling, and stimulating osteoclast differentiation and osteoclastic bone resorption
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